Specific Role of Chk1 Phosphorylations in Cell Survival and Checkpoint Activation

Abstract

Chk1 is a multifunctional protein kinase that plays essential roles in cell survival and cell cycle checkpoints. Chk1 is phosphorylated at multiple sites by several protein kinases, but the precise effects of these phosphorylations are largely unknown. Using a knockout-knockin system, we examined the abilities of Chk1 mutants to reverse the defects of Chk1-null cells. Wild-type Chk1 could rescue all the defects of Chk1-null cells. Like endogenous Chk1, wild-type Chk1 localized in both the cytoplasm and the nucleus, and its centrosomal association was enhanced by DNA damage. The mutation at S345 resulted in mitotic catastrophe, impaired checkpoints, and loss of the ability to localize in the cytoplasm, but the mutant retained the ability to be released from chromatin upon encountering genotoxic stressors. In contrast, the mutation at S317 resulted in impaired checkpoints and loss of chromatin release upon encountering genotoxic stressors, but its mutant retained the abilities to prevent mitotic catastrophes and to localize in the cytoplasm, suggesting the distinct effects of these phosphorylations. The forced immobilization of S317A/S345A in centrosomes resulted in the prevention of apoptosis in the presence or absence of DNA damage. Thus, two-step phosphorylation of Chk1 at S317 and S345 appeared to be required for proper localization of Chk1 to centrosomes.

Cdk-mediated phosphorylation of Chk1 is required for efficient activation and full checkpoint proficiency in response to DNA damage

“…Cdk phosphorylation of Chk1 S286/301 is required for optimal checkpoint proficiency ATR-mediated phosphorylation of S345 has been shown to be crucial for both biochemical activation of Chk1 and checkpoint proficiency (Niida et al, 2007;Walker et al, 2009). The finding that S345 phosphorylation of the Chk1 S2A mutant protein was impaired compared with wild-type after DNA damage raised the Cdk phosphorylation regulates Chk1 activation N Xu et al question of whether its biological function was similarly affected.…”

A nuclear budding mechanism in transiently arrested cells generates drug-sensitive and drug-resistant cells

“…Several DNA-binding drugs have been reported to produce mitotic catastrophe in both cells containing wild-type p53 and those bearing mutated or deleted p53 genes [13][14][15][16][17]. Mitotic catastrophe occurs in tumor cells treated with various antitumour drugs or radiation [7,13,14,[16][17][18][19][20][21]. These antitumour agents can also limit tumor growth through accelerated senescence arrest [10,12,13,22].…”

“…Senescence may also follow mitotic catastrophe if polyploid cells are arrested in G1 or G2 after mitotic catastrophe [13,24], but the arrest is not permanent when, for example, cells lack p53 function, or the p53 and p21 WAF1 protein levels decrease after treatment with certain drugs [7,11,13,21,22,25], or as a result of alterations in the phosphorilation of Chk1 [19]. Therefore, cells can re-enter the cell cycle, increasing their polyploidy before they eventually die.…”

Evaluation of CHK1 activation in vulvar squamous cell carcinoma and its potential as a therapeutic target in vitro

“…Another interesting observation is the high expression of pCHK1 Ser280 in normal vulvar epithelium. It has been shown that this phosphorylation can be mediated by Akt,31 RSK 13 in different cell types, possibly leading to cytoplasmic sequestration. Phosphorylation of this residue is not required for checkpoint function in untransformed cells 31.…”

“…It has been shown that this phosphorylation can be mediated by Akt,31 RSK 13 in different cell types, possibly leading to cytoplasmic sequestration. Phosphorylation of this residue is not required for checkpoint function in untransformed cells 31. For this reason, it is likely that in normal vulvar epithelium pCHK1 Ser280 rather functions in other aspects of the normal cell cycle progression.…”